This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This new, one-year project was focused on the preliminary steps of producing and analyzing the protein immunogens for a future pre-clinical proof-of-principle testing vaccine trial with challenge by P. knowlesi blood-stage parasites. Two important Reticulocyte Binding-Like (RBL) recombinant proteins, PkNBPxa and PkNBPxb, representing N-terminal adhesive domain regions of these RBLs were produced in the E. coli system for achieving the goal of testing their immunogenicity and efficacy in vivo using the rhesus macaque animal model. This animal model will allow the stringent evaluation of the potential of RBLs as malaria vaccine candidates for future testing in humans. The expression and purification of the PkNBPxa and PkNBPxb recombinant proteins was extremely challenging due to their biochemical composition. Despite codon optimization during the cloning process, these proteins were expressed in inclusion bodies and at very high concentrations, independent of culture media type, host cell or temperature. Development of these candidate vaccine antigens required adjustment of procedures and persistence to achieve solubility and refolding. Effort was also expended to write an NIH R21 proposal to continue this research and conduct the planned pre-clinical vaccine trial in rhesus monkeys. This NIH R21 proposal was reviewed successfully and will be funded, allowing this project to proceed to the next level of carrying out the pre-clinical vaccine trial. This project was also extremely valuable for supporting the productive Emory and CDC collaboration on this topic.